This invention relates to plasminogen activators which are useful thrombolytic agents. More particularly, this invention relates to glycosylated tissue plasminogen activator from cultured normal human colon cells.
It is known that various plasminogen activators (PA) are widely distributed throughout the body and can be purified from tissue extracts. Typical examples of tissue sources are kidney and lung tissues. The best characterized of these plasminogen activators fall into two major groups, urokinase plasminogen activator (u-PA) and tissue plasminogen activator (t-PA). u-PA and t-PA are present in ng/ml concentrations in human plasma but are immunologically unrelated. t-PA has been demonstrated to have higher affinity for fibrin than u-PA. u-PA products isolated and purified from human urine and from mammalian kidney cells are pharmaceutically available as thrombolytic agents.
Due to the extremely low concentration of t-PA in blood and tissue extracts, other sources and means of producing this preferred thrombolytic agent have been sought after.
One method of producing t-PA on a large scale comprises isolating the protein from the culture fluid of human melanoma cells grown under in vitro cell culture conditions. An established human melanoma cell line (Bowes) has been used for this purpose. See, for example, European Patent Application No. 41,766, published Dec. 16, 1981; Rijken and Collen, J. Biol. Chem. 256(13), 7035-7041 (1981); and Kluft et al., Adv. Biotech. Proc. 2, Alan R. Liss, Inc., 1983, pp. 97-110. The Bowes melanoma t-PA is a glycoprotein which has a molecular weight of about 68,000-70,000 daltons and a 527 amino acid structure with serine as the N-terminal. The melanoma t-PA exists as two chains, an A-chain and a B-chain. It also separates into two variants in the A-chain, known as types I and II, which differ by about M.sub.r 2000-3000. See Ramby et al., FEBS Lett. 146 (2), 289-292 (1982), and Wallen et al., Eur. J. Biochem. 132, 681-686 (1983). Type I is glycosylated at Asn-117, Asn-184 and Asn-448 whereas Type II is glycosylated only at Asn-117 and Asn-448 according to Pohl et al., Biochemistry 23, 3701-3707 (1984). A high mannose structure has been assigned to Asn- 117 whereas two complex carbohydrate structures are assigned to Asn-184 and Asn-448 by Pohl et al., "EMBO Workshop on Plasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985.
Genetic information from the Bowes melanoma cell line also has been embodied in E. coli by conventional recombinant DNA gene splicing methods to permit the production of the t-PA protein moiety by that microorganism. See, for example, UK Patent Application No. 2,119,804, published Nov. 23, 1983; Pennica et al., Nature 301, 214-221 (1983); and Vehar et al., Biotech. 2 (12), 1051-1057 (1984). Recombinant t-PA produced by the expression of Bowes melanoma genetic material in cultured mammalian cells has been administered to humans with some measure of effectiveness. See Collen et. al., Circulation 70(16), 1012-1017 (1984).
Notwithstanding the apparent utility of the t-PA derived from Bowes melanoma, the use of cancer cells or genetic information derived from cancer cells can raise uncertain drug regulatory problems in the therapeutic use of such materials. Thus, it is known that cancer cells (transformed cells) can produce human transforming growth factors. See, for example, Delarco and todaro, Proc. Natl. Acad. Sci. USA 75, 4001-4005 (1978), and Todaro et al., Ibid., 77, 5258-5261 (1980). Even the smallest amount of residual DNA from the cancer cells can be integrated into and expressed in the E. coli or genetically engineered mammalian cells, thereby raising the possibility of harmful effects if t-PA from such source is administered to the patient. Although the risks may be small by the judicious use of various purification techniques and appropriate monitoring of patients, it still would be preferable to use a t-PA that was not derived from cancer cells either directly or indirectly. The possible presence of viral genetic material or oncogene product can raise significant objections to the use of clinical material thus derived from transformed cells.
Moreover, the recombinant-derived t-PA produced in E. coli is non-glycosylated and contains only the protein moiety of t-PA. Althuogh the specific function of the carbohydrate moiety on t-PA has not been determined, it is known, in general, that glycosylation can cause certain differences in the protein of which the following are of biological interest: antigenicity, stability, solubility and tertiary structure. The carbohydrate side-chains also can affect the protein's half life and target it to receptors on the appropriate cells. See, for example, Delente, Trends in Biotech. 3 (9), 218 (1985), and Van Brunt, Biotechnol. 4, 835-839 (1986). The functional properties of carbohydrate-depleted t-PA are further discussed by Little, et al., Biochemistry 23, 6191-6195 (1984), and by Opdenakker et al., "EMBO workshop on Plasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985. The latter scientists report that enzymatic cleavage of carbohydrate side-chains from the melanoma (Bowes) derived t-PA by treatment with .alpha.-mannosidase causes an increase in the biologic activity of the modified t-PA.
Accordingly, the production of glycosylated t-PA from normal human cells on a large scale would be highly desirable. Cultured normal human cells have been used as a source of t-PA as can be seen from U.S. Pat. Nos. 4,335,215, 4,505,893, 4,537,860, and 4,550,080. Although various cell sources are mentioned in said patents, apparently only primary embryonic (or fetal) kidney, lung, foreskin, skin and small intestines (Flow Laboratories) or the AG1523 cell line were actually cultured for the production of t-PA according to the disclosures. Brouty-Boye et al., Biotech 2 (12), 1058-1062 (1984), also disclose the use of normal human embryonic lung cells for the production of t-PA. Rijken and Collen, J. Biol. Chem. 256 (13), 7035-7041 (1981), and Pohl et al., FEBS Lett. 168(1), 29-32 (1984), disclose the use of human uterine tissue as a t-PA source material. However, none of the foregoing disclosures on normal human cell-derived t-PA define the carbohydrate structures on the t-PA protein.
Production of glycosylated t-PA in non-human mammalian cells also is known. Thus, Kaufman et al., Mol. Cell. Biol. 5, 1750-1759 (1985), and European Patent Application No. 117,059, published Aug. 29, 1984, describe the use of Chinese hamster ovary cells and Browne et al., Gene 33, 279-284 (1985), describe the use of mouse L cells for such production. Kaufman et al., state that the Chinese hamster ovary t-PA is glycosylated in a similar but not identical manner as native t-PA. Glycosylated forms of t-PA obtained by recombinant DNA are further described by Zamarron et al., J. Biol. Chem. 259 (4), 2080-2083 (1984), and Collen et al., J. Pharmacol. Expertl. Therap. 231 (1), 146-152 (1984).
Production of glycosylated t-PA by recombinant DNA yeast cells also has been reported. Thus, European Patent Application No. 143,081, published May 29, 1985, describes a recombinant yeast plasmid vector which encodes human t-PA from Hela cells. The latter cells are known to be tumor derived. European Patent Application No. 174,835, published Mar. 19, 1986, describes a t-PA with selected glycosylation expressed in yeast. However, the cDNA encoding for the t-PA is derived from Bowes melanoma which also is tumor derived. European Patent Application No. 178,015, published Apr. 16, 1986, discloses a glycosylated uterine t-PA expressed in yeast cells or mouse cells. In the latter case, a bovine papilloma virus is used as the vector. Papilloma viruses have been implicated in natural cancers.
Although glycosylation is suggested in various of the foregoing European Patent Application disclosures, the glycosylation patterns are not described and sugar molecules are not identified.
It is apparent that differences in the glycosylation pattern on similar proteins can have profound effects on antigenicity, metabolism and other physiological properties. See, for example, the association of rheumatoid arthritis and osteoarthritis with changes in the glycocosylation pattern of total serum IgG by Parekh et al., Nature 316, 452-457 (1985).
Another example of a glycoprotein in which biological activity resides in the oligosaccharide moieties (i.e., particular structure at a specific site) is that of human chorionic gonadotropin (hCG). Thus, it is known that hCG without carbohydrate is a competitive inhibitor of native hCG; that oligo-saccharides isolated from hCG inhibit action of native hCG; and that tumor-produced hCG having the same amino acid sequence as native hCG but different sugars has almost no biological activity. See Calvo et al., Biochemistry 24, 1953-1959 (1985); Chen et al., J. Biol. Chem. 257 14446-14452 (1982).